RESEARCH ARTICLE Open Access

Stability of petal color polymorphism: thesignificance of anthocyanin accumulationin photosynthetic tissuesJosé Carlos Del Valle1* , Cristina Alcalde-Eon2, Mª. Teresa Escribano-Bailón2, Mª. Luisa Buide1,Justen B. Whittall3 and Eduardo Narbona1

Abstract

Background: Anthocyanins are the primary source of colour in flowers and also accumulate in vegetative tissues,where they have multiple protective roles traditionally attributed to early compounds of the metabolic pathway(flavonols, flavones, etc.). Petal-specific loss of anthocyanins in petals allows plants to escape from the negativepleiotropic effects of flavonoid and anthocyanins loss in vegetative organs, where they perform a plethora of essentialfunctions. Herein, we investigate the degree of pleiotropy at the biochemical scale in a pink-white flower colourpolymorphism in the shore campion, Silene littorea. We report the frequencies of pink and white individuals across 21populations and underlying biochemical profiles of three flower colour variants: anthocyanins present in all tissues(pink petals), petal-specific loss of anthocyanins (white petals), and loss of anthocyanins in all tissues (white petals).

Results: Individuals lacking anthocyanins only in petals represent a stable polymorphism in two populations at thenorthern edge of the species range (mean frequency 8–21%). Whereas, individuals lacking anthocyanins in the wholeplant were found across the species range, yet always at very low frequencies (< 1%). Biochemically, the flavonoidsdetected were anthocyanins and flavones; in pigmented individuals, concentrations of flavones were14–56× higher than anthocyanins across tissues with differences of > 100× detected in leaves. Loss of anthocyaninpigmentation, either in petals or in the whole plant, does not influence the ability of these phenotypes to synthesizeflavones, and this pattern was congruent among all sampled populations.

Conclusions: We found that all colour variants showed similar flavone profiles, either in petals or in the whole plant,and only the flower colour variant with anthocyanins in photosynthetic tissues persists as a stable flower colourpolymorphism. These findings suggest that anthocyanins in photosynthetic tissues, not flavonoid intermediates, are thetargets of non-pollinator mediated selection.

Keywords: Anthocyanins, Flavonoids, Flower color polymorphism, Loss of pigmentation, Non-pollinator mediatedselection, Plant secondary metabolites, Pleiotropy

BackgroundMutations are the primary source of genetic variation in allorganisms and have a key contribution to phenotypic diver-sity [1, 2], but not all mutations are evolutionarily relevant.Some phenotypic changes are produced through spontan-eous mutations with deleterious effects that are consistentlyeliminated by purifying selection [3]. In contrast, persistent

phenotypic changes arise from mutations maintained bybalancing selection through frequency-dependent or het-erogeneous selection or through the promotion of multipleadaptive peaks [4–8], resulting in a population polymorph-ism for that trait [9–11]. Factors that may determine whysome new phenotypes are fleeting and some persists aspolymorphisms are still open [12, 13], but the study offlower color is helping to shed light to this issue [14–17].Flower color variation has drawn the attention of

many naturalists through the history [18–20], and now-adays continues to be an important focus of research to

© The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

* Correspondence: jcdelgar@upo.es1Department of Molecular Biology and Biochemical Engineering, Pablo deOlavide University, 41013 Seville, SpainFull list of author information is available at the end of the article

Del Valle et al. BMC Plant Biology (2019) 19:496 https://doi.org/10.1186/s12870-019-2082-6

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evolutionary biologist [14, 21]. Flower color has beenconsidered as an adaptive trait for pollinator attraction[22], but underlying pigments also have other functions,especially in vegetative tissues. Anthocyanins are themost common plant pigment that color flowers, confer-ring orange, red, pink and blue colors [23] that attractdiverse functional groups of pollinators [22, 24]. Forexample, variation in anthocyanin content in monkey-flowers (Mimulus) results in red and pink-floweredspecies that are visited by hummingbirds and bees, re-spectively [25, 26]. In vegetative tissues, anthocyaninsmay show protective roles such as sunscreens, antioxi-dants or antipathogens, among others [27, 28]. Thus, lossof anthocyanins may affect pollinator activity, but mayalso have physiological effects depending on whether theyare accumulated or not in vegetative tissues. If the loss ofanthocyanins is confined to the flowers (usually in petals),the rest of the plant can produce anthocyanins and reduceany negative pleiotropic effects in other tissues [29, 30],whereas anthocyanins-lacking individuals in the wholeplant potentially grow and reproduce, but frequently ex-hibit fitness disadvantages that seem to explain their scar-city in the wild [31–33] (see Additional file 1: Table S1).Petal-specific loss of anthocyanins is frequently in-

duced by regulatory mutations (that is changes in theregulation of gene expression) and shows a mutationbias to Myb transcription factors, the key regulatory fac-tors controlling anthocyanin biosynthesis in plants [30,34]. Different copies of Myb proteins regulate floral andvegetative anthocyanins, thus the specificity of this regu-lation is predicted to have low pleiotropic consequences[35]. For example, in Ipomoea purpurea mutations theregulatory IpMyb1 gene are responsible for anthocyaninloss in pigmented flowers [36]. However, these muta-tions do not affect the fitness of white-flowered plantsand show equal or even higher reproductive successthan that of the pigmented individuals [15, 29].Anthocyanin-lacking individuals, on the other hand,

are conferred by loss-of-function in any of the structuralloci or whole plant regulatory genes of the anthocyaninbiosynthetic pathway (hereafter ABP) [30, 34]. Loss-of-function mutations may target a high spectrum of genessince there are more possible loci that could confer thenon-pigmented phenotype. Thus, the inactivation of anystructural gene of the pathway often limit the flux downthe ABP and block the anthocyanin production, but alsomay affect the synthesis of uncolored/pale-yellow non-anthocyanin flavonoids in the side branches of the path-way [23]. These flavonoids, such as flavones or flavonols,also perform important ecological functions becausethey show similar or even more protective functionsagainst environmental stressors than anthocyanins them-selves [37]. Therefore, the persistence of loss-of-functionmutations should be limited by the negative pleiotropic

effects associated to the absence of anthocyanins and/orintermediate non-anthocyanin flavonoids [14, 17, 38].The selection against these variants may depend onwhere the mutation occurs and the associated negativeconsequences for the flavonoid loss.Loss of pigmentation, particularly due to absence of

anthocyanins, represents the most frequent cases offlower color polymorphism in plants [39, 40]. White-flowered morphs represent valuable natural genotypes toknow the possible selective disadvantages of lack of antho-cyanins in the whole plant [33, 39, 41–43], but the non-anthocyanin flavonoid composition of such plants is un-known. In addition, the quantities of anthocyanins are usu-ally correlated with those of non-anthocyanin flavonoids,at least in some tissues, and the concentrations of the latterare even higher than that of anthocyanins [44, 45]. Conse-quently, it is difficult to distinguish which group of flavo-noids is responsible for the putative selective disadvantageof anthocyanin lacking plants [31–33], and studies thatclearly differentiate between flavonoid groups are limited.The shore campion Silene littorea Brot. (Caryophyllaceae)

is an annual pink-flowered species that accumulates antho-cyanins and non-anthocyanin flavonoids in petals and incalyces, leaves and stems [44, 46]. The accumulation ofboth kinds of flavonoids in vegetative tissues is highlyvariable and seems to respond to light stress [46]. Silene lit-torea grows along the Iberian coast, and exhibits ananthocyanin-based pink-white flower color polymorphismin two populations of the northwest distribution range, butanthocyanin-lacking individuals are occasionally observedin some populations [47]. In S. littorea, petal-specific poly-morphism is likely due to downregulation of the flavanone-3-hydroxylase (F3 h) gene through a downregulation of theSlMyb1a transcription factor [47], but genetic causes ofanthocyanin-lacking plants are still unknown. Flowers ofthis species are mainly visited by generalist pollinators fromthe orders Diptera, Hymenoptera, and Lepidoptera; how-ever, they do not seem to show strong pollinator preferencefor either pink or white flowers (M.L.B. 2019, unpublisheddata). Thus, the occurrence of petal anthocyanin loss andwhole-plant anthocyanin loss individuals in S. littorea(hereafter PAL and WAL phenotypes, respectively) offersan excellent opportunity for understanding the importanceof non-pollinator selection due to lack of anthocyaninsand/or non-anthocyanin flavonoids.In this study, we seek to understand the factors that de-

termine the fate of different forms of anthocyanin vari-ation in S. littorea. Thus, we investigated the populationfrequency of three anthocyanin phenotypes (PAL, WALand fully pigmented) in 21 populations across the speciesdistribution range over five years. Then, we used high-performance liquid chromatography coupled with diode-array detection and electrospray ionization tandem massspectrometry (HPLC-DAD-MSn) to study the flavonoid

Del Valle et al. BMC Plant Biology (2019) 19:496 Page 2 of 13

profiles at the whole plant level in the fully pigmentedphenotype and compare them to that of white-floweredvariants (i.e. PAL and WAL phenotypes); after that, thisstudy was expanded to more individuals and populationsusing spectrophotometric quantification of flavonoids. Be-cause of the negative consequences of the absence of fla-vonoids [30, 38], we expect the PAL phenotype to bemore common within a population compared to WAL.Thus, loss of anthocyanins and non-anthocyanin flavo-noids are expected to be limited to petals in PAL plants,but extended to the whole plant in WAL individuals.

ResultsFrequency of PAL, WAL and fully pigmented phenotypesOur population surveys confirmed that the PAL phenotypeis limited to two populations in the northern portion of thespecies range (Fig. 1). A polymorphism from 8 to 21% ofPAL plants has been maintained over the years at thesetwo populations (Additional file 2: Table S2). In contrast,WAL individuals were found in nine of the 21 populationssurveyed, including the two polymorphic populations, butalways at very low frequencies (< 1% of total plants; Fig. 1and Additional file 2: Table S2) and without any cleargeographic pattern.

Flavonoid identification and composition in each plant tissueFive anthocyanins and 21 flavones were identified inpetals, as well as four anthocyanins and 19 flavones inphotosynthetic tissues (Additional file 4: Table S3). The

anthocyanins detected were cyanidin derivatives in all cases,but with different substituents in petals and photosynthetictissues (Fig. 2 and Additional file 4: Table S3). The mainanthocyanin present in pigmented petals was a glycosylatedcyanidin with two sugars (one rhamnosylglucose and oneglucose) and acylated with acetic acid (representing 71.0–74.1% of the total anthocyanin concentrations; peak 3 inFig. 2c). In photosynthetic tissues, the structures of the pre-dominant anthocyanins were simpler, with only one sugarattached to the aglycone (78.0–99.4%; peaks 6–9 in Fig. 2g).The flavone composition was also different in petals

compared to photosynthetic tissues: isovitexin derivativeswere mostly accumulated in petals whereas isoorientin de-rivatives were the main flavones present in photosynthetictissues (Table 1 and Additional file 4: Table S3). The pri-mary petal flavone was an isovitexin glycosylated with twopentose sugars (69.5–88.3% of the total flavone concentra-tions in the three phenotypes; peak 17 in Fig. 2a and b).An isoorientin derivative containing an additional hexoseand a caffeoyl residue was the main flavone present incalyces (58.8–63.2%), leaves (42.9–58.1%) and stems(50.6–57.8%; peak 35 in Fig. 2e and f). In all tissues, iso-scoparin derivatives were also detected, but at relativelylow levels (< 11%; Table 1 and Additional file 4: Table S3).Flavones were the most abundant flavonoids detected

across tissues. In fully pigmented individuals, concentra-tions of flavones were 14–56× higher than anthocyaninsin petals, calyces and stems; leaf tissues showed an evergreat bias, producing flavones at rates >100x that of

Fig. 1 Silene littorea sampling and phenotypes with respect to anthocyanin accumulation. The map shows 21 populations covering the distributionrange of S. littorea where frequencies of petal anthocyanin loss (PAL) and whole-plant anthocyanin loss (WAL) phenotypes were estimated (seeAdditional file 2: Table S2). Pink circles indicate populations in which only fully pigmented individuals (pink petals and anthocyanic photosynthetictissues) are found, pink squares show populations in which WAL individuals are also found in at least one year of the studied period, and whitesquares represent populations where WAL and PAL individuals are found. The two polymorphic populations (Louro and Barra) and the two non-polymorphic populations (Trafalgar and Breña) in which flavonoids were biochemically analyzed are named. Photographs of the three phenotypespresent in polymorphic (above) and non-polymorphic populations (below) are presented. Note the differences in anthocyanin accumulation in calycesand stems in fully pigmented and PAL plants vs. WAL individuals (indicated by red arrows) (more photographs available in Additional file 3: Figure S1)

Del Valle et al. BMC Plant Biology (2019) 19:496 Page 3 of 13

Fig. 2 Examples of chromatograms of petals (a-d) and calyces (e-h) extracts from fully pigmented and WAL plants from Barra populationrecorded at 360 nm (flavones) and at 520 nm (anthocyanins). Only main peaks are numbered (details are shown in Additional file 5: Figure S2,Additional file 6: Figure S3 and Additional file 4: Table S3; A = phenolic acids)

Del Valle et al. BMC Plant Biology (2019) 19:496 Page 4 of 13

Table

1Meanconcen

tration(m

gg−

1FW

;fresh

weigh

t)of

maingrou

psof

anthocyanins

andflavone

sfro

mpe

talsandph

otosynthetictissues

offully

pigm

ented(F.P.),PA

Land

WALph

enotypes

ofBarraandBreñapo

pulatio

ns.Flavono

idqu

antificationwas

perfo

rmed

from

thearea

ofthepeaksde

tected

inthechromatog

ram

usingHPLC-DAD-M

Sn.Fou

rplantspe

rpheno

type

andpo

pulatio

nwereanalyzed.Flavonesweregrou

pedaccordingto

thefunctio

nalC

-glycosid

eflavone

core,the

additio

nalglycosestype

sandthe

hydroxycinnamicacid

typeslinkedto

flavone

skeleton

.Percentages

ofeach

flavono

idgrou

pspertotalflavonesareshow

ninparenthesesun

dercon

centratio

nvalues.A

ntho

cyanins

(cyanidinde

rivatives)sho

wed

similarchemicalcharacteristicsandweregrou

pedinto

asin

glecatego

ry.“–”

indicatesthat

thecompo

undwas

belowthedetectionlevel

Petals

Calyces

Leaves

Stem

s

Barra

Breñ

aBarra

Breñ

aBarra

Breñ

aBarra

Breñ

a

F.P.

PAL

WAL

F.P.

WAL

F.P.

PAL

WAL

F.P.

WAL

F.P.

PAL

WAL

F.P.

WAL

F.P.

PAL

WAL

F.P.

WAL

Antho

cyanins

Total:

1.65

0.03

0.02

2.82

0.07

0.28

0.35

<0.01

0.23

–<0.01

0.02

–0.05

–0.29

0.18

–0.29

–

Flavon

es

Total:

92.5

95.2

100.9

69.5

88.1

7.56

9.23

9.31

9.88

4.38

5.32

4.73

8.76

7.50

5.73

4.11

4.55

4.05

5.42

5.19

C-glycoside

flavone

core

a

Isovitexin

86.2

(93.1)

90.5

(95.1)

84.2

(83.4)

64.9

(93.3)

78.0

(88.5)

0.07

(0.96)

0.08

(0.83)

0.11

(1.15)

0.23

(2.35)

0.08

(1.91)

--

--

--

--

0.10

(1.84)

0.05

(0.90)

Isoscoparin

2.75

(2.98)

1.73

(1.81)

10.7

(10.6)

3.36

(4.84)

4.27

(4.85)

0.14

(1.89)

0.18

(1.97)

0.15

(1.66)

0.30

(3.06)

0.11

(2.57)

0.26

(4.95)

0.36

(7.55)

0.35

(3.96)

0.55

(7.33)

0.33

(5.74)

0.23

(5.71)

0.28

(6.21)

0.26

(6.39)

0.69

(12.7)

0.52

(10.0)

Isoo

rientin

1.49

(1.61)

0.96

(1.01)

3.53

(3.49)

0.73

(1.05)

3.28

(3.72)

6.62

(87.5)

7.95

(86.1)

8.25

(88.6)

8.47

(85.7)

3.69

(84.1)

4.76

(89.5)

3.68

(77.7)

7.90

(90.1)

6.48

(86.4)

5.01

(87.4)

3.67

(89.2)

3.88

(85.5)

3.49

(86.2)

4.30

(79.4)

4.02

(77.5)

di-C-glycoside

s2.10

(2.27)

2.00

(2.10)

2.52

(2.50)

0.53

(0.76)

2.57

(2.92)

0.73

(9.6)

1.02

(11.0)

0.80

(8.6)

0.88

(8.93)

0.50

(11.4)

0.29

(5.5)

0.70

(14.7)

0.52

(5.89)

0.47

(6.27)

0.40

(6.09)

0.21

(5.08)

0.38

(8.32)

0.30

(7.41)

0.33

(6.07)

0.60

(11.5)

Add

ition

alglycoses

a

Hexose

0.22

(0.24)

-0.77

(0.76)

1.54

(2.22)

3.22

(3.66)

7.52

(99.5)

9.17

(99.4)

9.26

(99.5)

9.18

(92.9)

4.00

(91.2)

5.31

(99.7)

4.72

(99.6)

8.73

(99.6)

7.43

(99.0)

5.68

(99.0)

4.08

(99.4)

4.50

(99.1)

4.00

(98.8)

5.38

(99.2)

5.11

(98.4)

Pentose

88.0

(95.1)

91.4

(96.0)

92.5

(91.7)

63.9

(92.0)

77.5

(88.0)

--

--

--

--

--

--

--

-

Hexoseandpe

ntose

2.04

(2.20)

1.65

(1.73)

3.27

(3.24)

2.98

(4.29)

3.85

(4.37)

--

--

--

--

--

--

--

-

Non

e2.25

(2.44)

2.17

(2.28)

4.36

(4.32)

1.06

(1.53)

3.53

(4.00)

0.04

(0.48)

0.06

(0.63)

0.05

(0.52)

0.70

(7.10)

0.38

(8.77)

0.01

(0.28)

0.02

(0.39)

0.04

(0.41)

0.07

(0.96)

0.06

(0.98)

0.02

(0.60)

0.04

(0.95)

0.05

(1.23)

0.04

(0.80)

0.08

(1.56)

Acylatio

na

Acylated

0.74

(0.80)

1.07

(1.12)

1.91

(1.90)

1.58

(2.27)

2.47

(2.80)

6.82

(90.3)

8.08

(87.6)

8.35

(89.7)

8.22

(83.2)

3.55

(81.0)

5.21

(97.9)

4.50

(95.1)

8.59

(98.0)

7.29

(97.1)

5.53

(96.5)

3.90

(95.0)

4.23

(93.0)

3.85

(95.3)

5.31

(97.9)

4.92

(94.8)

Caffeicac.

0.54

(0.58)

0.89

(0.93)

1.11

(1.10)

0.29

(0.42)

0.52

(0.59)

5.37

(71.1)

6.46

(70.1)

6.80

(73.1)

6.14

(62.1)

2.66

(60.7)

3.71

(69.8)

3.15

(66.5)

6.36

(72.6)

4.50

(60.0)

3.37

(58.7)

3.01

(73.2)

3.28

(72.2)

3.02

(74.7)

2.91

(53.8)

2.65

(51.1)

Ferulic

ac.

0.20

(0.22)

0.18

(0.19)

0.81

(0.80)

1.29

(1.85)

1.95

(2.21)

1.38

(18.2)

1.51

(16.3)

1.46

(15.7)

1.48

(15.0)

0.57

(13.0)

1.39

(26.1)

1.16

(24.6)

1.86

(21.3)

2.03

(27.1)

1.35

(23.5)

0.84

(20.5)

0.83

(18.2)

0.74

(18.3)

0.76

(14.0)

0.81

(15.7)

p-coum

aricac.

--

--

--

--

--

--

--

--

--

1.15

(21.2)

0.79

(15.2)

Diacylated

--

--

-0.07

(0.96)

0.11

(1.22)

0.09

(0.96)

0.61

(6.14)

0.32

(7.35)

0.11

(2.01)

0.19

(3.98)

0.36

(4.16)

0.76

(10.1)

0.82

(14.2)

0.05

(1.28)

0.12

(2.57)

0.09

(2.26)

0.49

(8.96)

0.67

(12.8)

Non

e91.8

(99.2)

94.2

(98.4)

99.0

(98.1)

67.9

(97.7)

85.7

(97.2)

0.74

(9.74)

1.14

(12.4)

0.96

(10.3)

1.66

(16.8)

0.83

(19.0)

0.11

(2.08)

0.23

(4.93)

0.18

(2.02)

0.22

(2.91)

0.20

(3.51)

0.21

(5.02)

0.32

(7.00)

0.19

(4.72)

0.11

(2.05)

0.27

(5.17)

a che

mical

characteristicsof

flavo

noidsarede

tailedin

Add

ition

alfile4:

TableS3

Del Valle et al. BMC Plant Biology (2019) 19:496 Page 5 of 13

anthocyanins (Table 1). These differences were even moreapparent in white petals of PAL plants and anthocyanin-lacking WAL individuals.

Variation in flavonoid content among phenotypes usingHPLC-DAD-MSn

The three phenotypes showed significant differences intheir anthocyanin concentrations. In petals, PAL andWAL phenotypes accumulated only 1% of the same an-thocyanins found in the fully pigmented phenotype (Fig.2d; Tables 1 and 2). In calyces and stems, WAL pheno-types produced undetectable concentration of anthocya-nins (Fig. 2h), whereas fully pigmented and PALphenotypes showed similar anthocyanin levels. In leaves,anthocyanin concentration was very low and statisticallysimilar for the three phenotypes.In contrast to the differences found in anthocyanin

concentrations, the three phenotypes showed minimaldifferences in their flavone content (Fig. 2a, b, e and f),and only three petal flavones (~ 1.5% of total flavones)were not present in all phenotypes (see Additional file 4:Table S3). We found differences in flavone compositionbetween the polymorphic and non-polymorphic popula-tions, with five compounds specific to Breña (com-pounds 16, 18, 21, 33a and 37a in Additional file 4:Table S3). The first three compounds were rare non-acylated O-glycosyl-C-monoglycoside flavones of petals(< 1% of total flavones), whereas the other two weremoderately abundant in photosynthetic tissues (4.17–21.2% of total flavones). Thus, PCA based on the flavonecomposition and concentration showed higher separ-ation between populations than among phenotypes ofeach population (Additional file 7: Figure S4). When wecompared the flavone concentration of each specific

group of flavones (i.e. derivatives of isovitexin, isoor-ientin and isoscoparin, and di-C-glycosides), we foundno significant difference among phenotypes neither inthe polymorphic nor the non-polymorphic population(Table 2). Similar results were obtained when usingthe relative proportion of flavones in MANOVAs(Additional file 8: Table S4).

Variation in flavonoid content among phenotypesmeasured spectrophotometricallyWhen expanding the sampling to more individuals andpopulations, spectrophotometric quantification showedsimilar pattern of anthocyanin and flavone production tothat found in the HPLC analysis. In the two polymorphicpopulations, the three phenotypes showed significantdifferences for the anthocyanin accumulation in all tis-sues except for leaves, which showed very low values inall phenotypes (Fig. 3; Table 3). In petals, PAL and WALphenotypes produced near zero anthocyanin concentra-tion, whereas in photosynthetic tissues only the WALphenotype lacked anthocyanins. The three phenotypesshowed statistically similar flavone concentrations inphotosynthetic tissues. In petals, significant differenceswere found due to the higher flavone content in WALplants from Barra (Fig. 3). Between populations, signifi-cant differences were found for the anthocyanin produc-tion in petals and stems, and for the flavone productionin all tissues. Anthocyanins and flavones concentrationswere, in general, higher in Barra than Louro.In non-polymorphic populations, anthocyanin produc-

tion in petals and calyces were significantly different be-tween fully pigmented and WAL phenotypes, and nearsignificant in stems (Fig. 3; Table 3). Flavone concentra-tions in both phenotypes were similar in all tissues

Table 2 Results from ANOVAs and MANOVAs comparing the anthocyanin and flavone contents among phenotypes in Barra (fullypigmented, PAL and WAL) and Breña (fully pigmented and WAL). Anthocyanin and flavone concentrations were obtained fromHPLC analyses performed in four plants of each phenotype. Comparisons were made independently for each plant tissue. Totalanthocyanins were considered for anthocyanin analyses, whereas main groups according to the C-glycoside flavone core wereconsidered for flavone analyses (see Table 1)

Tissue Anthocyanins Flavones

ANOVA test MANOVA test

SS d.f. F P Wilk’s lambda F d.f. P

Barra Petals 6.403 2, 12 19.32 0.001a 0.215 1.735 4, 12 0.188

Calyces 0.272 2, 12 8.249 0.009b 0.563 0.500 4, 12 0.834

Leaves 0.001 2, 12 2.627 0.126 0.256 1.463 4, 12 0.266

Stems 0.173 2, 12 17.28 < 0.001b 0.248 1.510 4, 12 0.251

Breña Petals 15.11 1, 8 49.89 < 0.001 0.659 0.389 4, 8 0.808

Calyces 0.106 1, 8 30.76 < 0.001 0.260 2.131 4, 8 0.280

Leaves 0.004 1, 8 1.121 0.330 0.445 0.935 4, 8 0.544

Stems 0.170 1, 8 10.71 0.017 0.121 5.448 4, 8 0.098apost hoc Tukey test showed significant differences between fully pigmented vs. PAL and WAL phenotypes (P < 0.05)bpost hoc Tukey test showed significant differences between WAL vs. fully pigmented and PAL phenotypes (P < 0.05)

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except for calyces, in which WAL plants from Breñashowed nearly half concentrations compared to fully pig-mented plants (Fig. 3). Between populations, significant dif-ferences were found for anthocyanins in all tissues exceptfor the stems, and for flavones in all photosynthetic tissues,showing higher concentration levels in Breña population.

DiscussionIn this study, we found that shore campion accumulatesboth anthocyanins and flavones, but specific classes ofthese compounds were differentially produced in petalsversus photosynthetic calyces, leaves and stems. Fullypigmented and PAL plants showed similar anthocyanincontent in the analyzed tissues, except for the obviousabsence in petals, whereas WAL phenotype lacks antho-cyanins in the whole plant. In contrast, plants with whitepetals (both PAL and WAL phenotypes) have similar fla-vone composition and concentration compared to pink-flowered plants. Thus, the synthesis of flavones in eachtissue of both PAL and WAL phenotypes seems to benot influenced by the loss of anthocyanins. This patternof anthocyanin and flavone production in all phenotypeswas congruent in the distant polymorphic and non-

polymorphic populations. Together, these results suggestthat anthocyanin accumulation in photosynthetic tissuesare directly or indirectly involved in petal color poly-morphism persistence. Below, we discuss these findingsin view of the frequency in which PAL and WAL pheno-types are found in natural populations.One of the most significant findings reported here is

that PAL and WAL plants exhibited similar flavone con-tent as fully pigmented plants, even though they lackedanthocyanins in either their petals or petals and vegeta-tive tissues. In a previous study analyzing the sequencesand gene expression of ABP genes in petals of S. littorea,Casimiro-Soriguer et al. [47] suggested that anthocyaninpetal-loss in PAL individuals is caused by a decreasedexpression of flavanone-3-hydroxylase (F3h) controlledby a petal specific regulatory gene, SlMyb1a. Detectionof flavones in PAL petals is consistent with the blockageof the ABP at F3 h since flavones are synthetized fromnaringenin or eriodictyol, which are produced in thesteps immediately preceding F3H (Fig. 4). We suggestthat downregulation of F3 h prevents a blockage of fla-vone production [48] and redirects flux from anthocya-nins to flavones in white petals of S. littorea, as is

Fig. 3 Flavonoid concentrations measured by spectrophotometry in the phenotypes of S. littorea from the polymorphic (Barra and Louro) andnon-polymorphic (Trafalgar and Breña) petal-color populations. Mean (± s.e.) concentrations of anthocyanins and non-anthocyanin flavonoids inthe four studied plant tissues are showed. Pink, pink-white striped and white bars represent fully pigmented, PAL and WAL phenotypes,respectively. Letters indicate significant differences (P < 0.05) from post hoc multiple comparisons among phenotypes within each population.Note the different scale between plant tissues and flavonoid types. FW, fresh weight

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described in other flower color polymorphic species[49, 50]. Similarly, mutations leading to the completelack of anthocyanins in WAL individuals may occurlater in the pathway to preserve flavone production.In Mimulus lewisii and Iochroma calycinum, mutations incoding regions of a late gene of the ABP, the dihydroflavo-nol 4-reductase (Dfr), cause the complete loss of anthocy-anins of rare white-flowered individuals [51, 52]. A similardownstream blockage of the ABP, but not necessarilycaused by inactivation of DFR, could explain the absenceof anthocyanins in WAL individuals.Although several studies have examined the genetic

and molecular basis for PAL and WAL phenotypes[11, 29, 48, 51–53], this is the first study that hascompared the complete flavonoid profile in these twoforms of white-flowered individuals. Nevertheless, somestudies have approached this goal in wild species. For ex-ample, flowers of the rare white-petal phenotype ofIochroma calycinum accumulate the same flavonol (quer-cetin) that the pigmented phenotype as determined bythin layer chromatography [52]. Although comparisonsbetween natural and horticultural plants should be ad-dressed carefully since they are under different selectionwhen in cultivation, there is some biochemical knowledgeavailable for ornamental plants. Several studies have re-ported similar flavonol profiles for white-flowered andpigmented lines, as for lisianthus (Eustoma grandiflorum)

and gentians (Gentiana triflora) [54, 55]; however, somewhite-flowered lines showed different flavonoid profiles,probably because of distinct genetic blockage of the ABP.We have found that all WAL plants of the shore campionshowed the same flavonoid profile. This lack of flavonoiddiversity in WAL phenotype reinforces the assumptionthat loss-of-function mutations may target specific lategenes rather than the early genes of the ABP, which wouldcompromise flavone production. The shore campion doesnot produce others classes of flavonoids others than fla-vones and anthocyanins [47]. Thus, any mutation affectingan ABP gene prior to F3 h should preclude any flavoneproduction (Fig. 4), making this mutation selectively dis-advantageous due to the decisive role of these compoundsto plant development and survival [28, 37, 56].Loss of anthocyanin pigments is relatively common in

nature [39, 40, 57], but its effects on plant fitness will de-termine the fate of white-flowered individuals. In severalspecies, PAL phenotypes generally show similar, or evenhigher, fitness than fully pigmented plants ([40, 58, 59];but see, for instance, [11]), resulting in stable flower colorpolymorphism in populations. In the shore campion, thepink-white polymorphism is maintained over the years,and white flowers represents 8–21% of total plants in thetwo polymorphic populations. Myb-mediated loss of an-thocyanins, as for S. littorea, are frequently cell or tissuespecific [60, 61] and allow downregulation of petal

Table 3 Results from generalized linear models (GLMs) testing differences among phenotypes, populations and their interaction onthe production of total anthocyanins and non-anthocyanin flavonoids in each plant tissue. GLMs were performed separately inpolymorphic (Barra and Louro) and non-polymorphic populations (Breña and Trafalgar). Anthocyanin and flavone concentrationswere obtained from spectrophotometric quantification of flavonoids

Source ofvariation

Polymorphic populations Non-polymorphic populations

Anthocyanins Flavones Anthocyanins Flavones

d.f. F P d.f. F P d.f. F P d.f. F P

Petals

Phenotype 2 1447.9 < 0.001 2 4.122 0.020 1 74.58 < 0.001 1 1.284 0.269

Population 1 6.450 0.013 1 4.510 0.036 1 11.54 0.002 1 0.644 0.430

Phen. x Pop. 2 2.525 0.086 2 1.229 0.298 1 2.939 0.100 1 0.945 0.341

Calyces

Phenotype 2 33.47 < 0.001 2 1.019 0.366 1 27.67 < 0.001 1 10.27 0.004

Population 1 0.368 0.546 1 35.15 < 0.001 1 8.231 0.008 1 20.10 < 0.001

Phen. x Pop. 2 0.314 0.732 2 0.688 0.506 1 3.475 0.075 1 1.785 0.194

Leaves

Phenotype 2 1.512 0.227 2 1.407 0.251 1 2.864 0.104 1 0.032 0.860

Population 1 0.001 0.990 1 10.48 0.002 1 4.672 0.041 1 35.24 < 0.001

Phen. x Pop. 2 0.422 0.657 2 2.862 0.063 1 1.391 0.250 1 1.366 0.254

Stems

Phenotype 2 25.28 < 0.001 2 2.285 0.109 1 3.266 0.084 1 1.994 0.172

Population 1 27.78 < 0.001 1 15.49 < 0.001 1 0.157 0.696 1 11.98 0.002

Phen. x Pop. 2 7.131 0.001 2 0.101 0.904 1 0.042 0.840 1 8.106 0.009

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anthocyanins without hindering anthocyanin accumula-tion in other tissues. Our biochemical results confirmedthat PAL and fully pigmented plants have similar antho-cyanin content in photosynthetic tissues, in addition tohave similar flavone content, which is expected to havefew or no pleiotropic effects that could alter fitness ofPAL phenotypes [17, 34]. In fact, snails and caterpillarsproduced similar herbivory levels in leaves of fully pig-mented and PAL plants of Barra, but white petals of PALplants showed higher hemipteran florivory that petals offully pigmented plants (M.L.B. 2019, unpublished data).A key question arising from our findings is, “Why are

WAL phenotypes so rare even if they have similar amountsof protective flavones than fully pigmented plants?”. Fla-vones share many of the numerous protective biologicalfunctions attributed to anthocyanins [27, 56, 62], and are atleast 14 times more abundant than anthocyanins across tis-sues of the shore campion, as commonly found in otherspecies [63]. In S. littorea, petal flavones (isovitexins) couldbe involved in regulation of vacuole homeostasis in epider-mal cells and/or act as co-pigments of anthocyanins [64,65], whereas flavones accumulated in photosynthetic tis-sues (isoorientins) are effective antioxidants that may playimportant functions in stress tolerance [28, 62]. Since theseprotective flavones are accumulated in reproductive andvegetative tissues of WAL plants, it seems plausible thatthe loss of anthocyanins could be involved in the ecological

disadvantage by which WAL phenotype remains scarce inthe populations. In fact, it is recently proposed that antho-cyanins may play a decisive role in the regulation ofsignaling cascades responsible for cell growth and differen-tiation; thus, controlling important developmental pro-cesses [27, 28, 66, 67]. In addition, genetic linkage betweenABP genes and other loci affecting fitness [16], as well asthe metabolic cross-talk between flavonoid and othermetabolic pathways [68], are other possible explanationsfor why loss of anthocyanins (not the flavonoid intermedi-ates) seems to restrict the spread of WAL phenotypes.Taken together, our results suggest that the ability to pro-duce anthocyanin pigments in photosynthetic tissues ofthe shore campion is associated with the ability to generatestable petal color polymorphisms.

ConclusionsIn summary, our results show striking differences in theability to synthesize anthocyanins between fully pigmentedand white-petal variants of S. littorea, whereas flavoneproduction is not affected by loss of anthocyanin pig-ments. Differences in flavonoid profile between PAL andWAL individuals are based in the absence of anthocyaninsin petals or the whole plant, respectively. The low fre-quency of WAL plants in natural populations leads us toconsider the negative ecological consequences of antho-cyanin loss, or other putative pleiotropically-linked traits,

Fig. 4 Simplified flavonoid biosynthetic pathway in Silene littorea. Enzymatic activities (capital letters next to arrows) and metabolic products areindicated. Main anthocyanins and flavones detected by HPLC-DAD-MSn are in boxes with red, green and yellow letters for compounds found inpetals, photosynthetic tissues or both, respectively. The biosynthetic route was divided into early and late halves using a dotted line based ongenes involved in the synthesis of upstream (flavones) and downstream (anthocyanins) products of the biosynthetic pathway. CHS: chalconesynthase; CHI, chalcone isomerase; F3’H, flavonoid 3’hydroxylase; FNS, flavone synthase; F3H, flavanone-3-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; GT, glucosyl transferase; RT, rhamnosyl transferase; AT, acyltransferase

Del Valle et al. BMC Plant Biology (2019) 19:496 Page 9 of 13

in photosynthetic tissues and suggests the critical role ofthese compounds to generate stable flower color poly-morphism. It will be interesting to analyze the flavonoidprofile in other species with anthocyanin loss individualseither in the petals or in whole plant, and linking this in-formation with their population frequency. These data willprovide new insights into the flower color evolution,which also may produce new knowledge on microevolu-tionary processes.

MethodsStudy species and frequency of anthocyanin-deficientphenotypesSilene littorea is an annual wild plant that grows incoastal dune ecosystems from the northwestern to thesoutheastern Iberian Peninsula [47]. Depending on the de-gree of human disturbance on their habitat, population’ssize ranges from approx. 100 (e.g. Algezur, Odiel) to morethan 10,000 individuals (e.g. Sines, Barra). This self-compatible species is entomophilous, but may produce upto 20% of fruit and seed set by spontaneous autogamy[69]. Calyces, leaves and stems produce chlorophyllsshowing photosynthetic activity [70].To account for the population frequency of PAL and

WAL phenotypes, we sampled 21 populations coveringthe full distribution range of S. littorea (Fig. 1). In popula-tions with more than 1000 estimated individuals, fre-quency estimates were carried out following a randomsampling; in populations with lower number of individ-uals, the whole population was carefully sampled. Plantswere visually categorized to each phenotype based on theabsence or presence of anthocyanins in petals and photo-synthetic tissues. PAL and WAL individuals showed asimilar morphology (plant height, number of open flowers,flower size) than the fully pigmented phenotype.

Sampling for flavonoid analysesFlavonoid analyses were conducted in two northwesternpetal-color polymorphic populations (Barra and Louro)and in two southern non-polymorphic populations(Breña and Trafalgar; Fig. 1). For complete flavonoididentification, we randomly selected four plants of eachfully pigmented, PAL and WAL phenotypes in Barra andfour plants from each fully pigmented and WAL pheno-types in Breña. For spectrophotometric flavonoids quan-tification in these four populations, we extended thesampling to 11–20 plants of each phenotype except forthe rare WAL individuals, in which six, five, six andthree individuals were analyzed (Barra, Louro, Breña andTrafalgar, respectively). Sampling was carried out fromMarch to April 2016, during the early to mid-floweringperiod of the species (permissions were not necessary tocollect these samples). For each plant, we collected foursamples: the petals and calyx of one flower, a leaf and

stem sections (1 cm length) from the middle region of thestem. Flavonoids were extracted in 1.5ml of MeOH con-taining 1% HCl and stored at − 20 °C in the dark, followingthe procedure described in Del Valle et al. [44]. Voucherspecimens from these populations are deposited at thePablo de Olavide University Herbarium (UPOS-3954,UPOS-8982, UPOS-8983 and UPOS-8984).

Flavonoid identification and quantification by HPLC-DAD-MSn

A volume of 500 μL of methanolic extracts of each planttissue (i.e. petals, calyces, leaves and stems) was concen-trated in a SpeedVac concentrator (Savant ISS110,Thermo Fisher Scientific, NC, USA) after the addition of100 μL of ultrapure water (Autwomatic, Wasserlab, Bar-batáin, Spain). The volume of the aqueous extracts wasthen adjusted to 250 μL with acidified water (pH = 1.4,HCl). Aqueous extracts were filtered (Clarinert™ SyringFilters, 0.45 μm, Agela Technologies, DE, USA) prior tothe HPLC-DAD-MSn analysis. We followed the proced-ure described in Alcalde-Eon et al. [71, 72], which hasprovided satisfactory results in the analyses of anthocya-nins in other plant materials and also allows the simul-taneous detection of anthocyanins and flavones (seeresults) in a single run. HPLC analyses were carried outin a Hewlett-Packard 1100 series liquid chromatograph(Agilent Technologies, Waldbronn, Germany). Detectionwas carried out at 360 nm and 520 nm for flavone andanthocyanin analysis, respectively. Spectra were recordedfrom 220 to 600 nm.Mass spectrometric analyses were performed in an API

3200 Qtrap equipped with an ESI source and a triplequadrupole-ion trap mass analyzer that was controlled byAnalyst v.5.1 software (Applied Biosystems, Darmstadt,Germany). The HPLC system was connected to the massspectrometer via the UV cell outlet. Positive mode (ESI+)and specific conditions were selected to allow the simul-taneous detection of anthocyanins and flavones (see Add-itional file 9: Supplementary methods for details).Identification of the compounds was done considering

their retention times, UV-Vis spectra, m/z of the molecu-lar ion (M+) for anthocyanins or m/z of the protoned ion[M+H]+ for flavones, fragment ions and fragmentationpatterns of the compounds. These data supplied valuableinformation concerning the nature of the aglycones andsubstituents, which was further compared to the featuresof standards reported in the literature [73] and to thosestandards (cyanidin 3-O-glucoside, isovitexin (apigenin 6-C-glucoside) and isoorientin (luteolin 6-C-glucoside)) andsamples with known composition on barley (Hordeumvulgare L. [74]) analyzed in the same conditions. Further-more, alkaline and acid hydrolyses were performed in allthe types of sample extracts (petals and vegetative parts)to verify the presence of acids in the molecules as well as

Del Valle et al. BMC Plant Biology (2019) 19:496 Page 10 of 13

to determine their identity (see Additional file 9: Supple-mentary methods for details). Some of the major com-pounds of petals and photosynthetic tissues were isolatedand alkaline hydrolysis was also carried out on them.Anthocyanin quantification was done from the area of

the peaks detected in the chromatogram recorded at520 nm and using a Cyanidin 3-O-glucoside (Polyphe-nols Labs, Sandnes, Norway) calibration curve. Likewise,flavone quantification was done from the area of thepeaks detected in the chromatogram recorded at 360nm. A calibration curve of isovitexin and isoorientin(Extrasynthese, Genay, France) was employed to quantifythe flavones present in petals and photosynthetic tissues,respectively.HPLC results were analyzed separately for flavones

and anthocyanins. In each tissue, exploratory analyses offlavone content of the different phenotypes were per-formed by principal-component analysis (PCA), usingconcentrations of all compounds identified. We retainedthose compounds with the highest principal componentloadings. In addition, compounds highly correlated wereeliminated to overcome co-linearity. PCA was based onthe covariance matrix and without rotation of the ex-tracted component [75]. Confirmatory MANOVAs(multivariate analysis of variance) were carried out whendifferences between populations were detected [75].MANOVAs were performed grouping the total flavonecomposition of plants into four functional groups ac-cording to the C-glycoside flavone core (i.e. derivativesof isovitexin, isoorientin and isoscoparin, and di-C-glycosides; see Table 1). Since plants from Barra andBreña locations showed differences in their flavone con-tent (see results) and the PAL phenotype is only presentin Barra, both populations were analyzed independently.Because only a few anthocyanin compounds were foundin the samples and some phenotypes did not produceanthocyanins (see results), exploratory PCAs could notbe performed; instead, differences in the total anthocya-nin concentrations among phenotypes were analyzedusing ANOVAs with post hoc Tukey test. ANOVAs,PCAs and MANOVAs were carried out in SPSS v.22.0(Armonk, NY, IBM Corp.).

Spectrophotometric flavonoids quantificationWe used a Multiskan GO microplate spectrophotometer(Thermo Fisher Scientific Inc., MA, USA) to quantifyglobal anthocyanin and flavone concentrations in orderto expand the study to more individuals and popula-tions. Three replicas of 200 μL were measured for ofeach tissues of all individuals. Absorbances were read at350 and 520 nm to determine the concentrations of fla-vones and anthocyanins respectively [46], and their con-centrations were calculated using calibration curves ofstandards of the main compounds found (i.e. cyanidin 3-

O-glucoside, isovitexin and isoorientin) and expressed ascyanidin-3-glucoside, isovitexin and isoorientin equiva-lents in fresh weight, respectively.Generalized linear models (GLMs) with Gaussian or

gamma error distribution were performed in R v3.4.0 [76]to test for differences in the accumulation of anthocyaninsand flavones in each plant tissue; phenotype and popula-tion were considered as fixed factors. Previously, we testedthe error distributions that generated the smaller deviancein the model, using the Akaike’s Information Criterion. F-test for analysis of deviance was used to correct foroverdispersion [77]. Multiple post hoc comparisons wereperformed using the “multcomp” R-package [78]. Sepa-rated GLMs were performed for polymorphic and non-polymorphic populations, using post hoc comparisonswith Bonferroni adjustment to test for differences amongphenotypes within each population.

Supplementary informationSupplementary information accompanies this paper at https://doi.org/10.1186/s12870-019-2082-6.

Additional file 1: Table S1. Frequency of petal anthocyanin loss (PAL)and whole-plant anthocyanin loss (WAL) individuals in natural populationsof species with polymorphism caused by loss of anthocyanins in petal andspontaneous white mutants, respectively.

Additional file 2: Table S2. Sites, geographical locations, estimatedpopulation size and percentage of petal anthocyanin loss (PAL) andwhole-plant anthocyanin loss (WAL) phenotypes of S. littorea.

Additional file 3: Figure S1. Pictures of white-flowered plants of S.littorea in which differences in anthocyanin accumulation in photosyn-thetic tissues between the petal anthocyanin loss (PAL) and whole-plantanthocyanin loss (WAL) phenotypes are shown.

Additional file 4: Table S3. Anthocyanins and flavones identifiedthrough HPLC-DAD-MSn from methanolic extracts of petals andphotosynthetic tissues (calyces, leaves and stems) of S. littorea plants.

Additional file 5: Figure S2. Chromatogram of the petal extract of aWAL specimen from Breña population recorded at 360 nm.

Additional file 6: Figure S3. Chromatogram of the leaf extract of afully pigmented specimen from Barra population recorded at 360 nm.

Additional file 7: Figure S4. Scatter plot of principal componentsextracted from PCAs using flavone composition detected in petals, calyces,leaves and stems of S. littorea phenotypes through HPLC-DAD-MSn.

Additional file 8: Table S4. Results from MANOVAs comparing therelative proportion of flavones among phenotypes in Barra (fullypigmented, PAL and WAL) and Breña (fully pigmented and WAL).

Additional file 9. Supplementary methods. Details of the massspectrometry conditions, isolation of flavones, and alkaline and acidhydrolysis.

AbbreviationsABP: Anthocyanin biosynthetic pathway; GLM: Generalized linear models;HPLC-DAD-MSn: High-performance liquid chromatography coupled withdiode-array detection and electrospray ionization tandem mass spectrometry;PAL: Petal anthocyanin loss; WAL: Whole-plant anthocyanin

AcknowledgementsThe authors thank Ágata Cardoso, Rebeca Ferreras and Antonio Gallardo forfield and laboratory assistance. We also thank reviewers for their usefulcomments and suggestions.

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https://doi.org/10.1186/s12870-019-2082-6https://doi.org/10.1186/s12870-019-2082-6

Authors’ contributionsEN, MLB and JBW conceived the ideas and designed the experiment; JCD,CAE and MTEB collected plant material and performed biochemical analyses;JCD, EN, JBW and CAE wrote the article. JCD carried out the speciesidentification in the field. All authors contributed to the manuscriptpreparation and gave final approval for publication.

FundingFinancial support was provided by the Spanish Government MINECOprojects (CGL2012–37646 and CGL2015–63827-P) and also through aPredoctoral Training Program grant to JCV (BES-2013–062610).

Availability of data and materialsThe datasets supporting the conclusions of this article are included withinthe article and its additional files.

Ethics approval and consent to participateNot applicable.

Consent for publicationNot applicable.

Competing interestsThe authors declare that they have no competing interests.

Author details1Department of Molecular Biology and Biochemical Engineering, Pablo deOlavide University, 41013 Seville, Spain. 2Grupo de Investigación enPolifenoles (GIP), University of Salamanca, 37007 Salamanca, Spain.3Department of Biology, Santa Clara University, Santa Clara, CA 95053, USA.

Received: 28 March 2019 Accepted: 17 October 2019

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AbstractBackgroundResultsConclusions

BackgroundResultsFrequency of PAL, WAL and fully pigmented phenotypesFlavonoid identification and composition in each plant tissueVariation in flavonoid content among phenotypes using HPLC-DAD-MSnVariation in flavonoid content among phenotypes measured spectrophotometrically

DiscussionConclusionsMethodsStudy species and frequency of anthocyanin-deficient phenotypesSampling for flavonoid analysesFlavonoid identification and quantification by HPLC-DAD-MSnSpectrophotometric flavonoids quantification

Supplementary informationAbbreviationsAcknowledgementsAuthors’ contributionsFundingAvailability of data and materialsEthics approval and consent to participateConsent for publicationCompeting interestsAuthor detailsReferencesPublisher’s Note